| Background Zinc finger protein which regulates apoptosis and cell cycle arrest(ZAC) gene is a tumor suppressor gene, located in human chromosome 6q24-25. The protein,encoded by ZAC gene, is a transcription factor with 7 zinc finger domains, which can induce cell apoptosis and cell cycle Gl arrest, thus inhibiting the growth of tumor cells. p300 is a t ranscription cofactor with histone acetyltransferase(HAT) activity,which can acetylate all 4 core histones and 70 other proteins at multiple residues.Our previous studies showed that somatostatin analogues octreotide(OCT) could upregulate the expression of ZAC in gastric cancer cells. ZAC interacted with p300 and increased the activity of p300-HAT, and significantly increased lysine 14 acetylation of histone H3.Objective To observe whether p300 and OCT have a synergistic effect on inhibiting the proliferation of gastric cancer cells; whether p300 can upregulate the expression of ZAC gene in gastric cancer cells.Materials and methods1. Identification of eukaryotic expression vector of wild type p300 Wild type p300 plasmid was transfected into E.coli DH5α. The plasmid DNA was and extracted and identified by agarose gel electrophoresis and sequencing.2. Experimental groups The gastric cancer cells SGC7901 and BGC823 were randomly divided into 4 groups: experimental group 1: transfected with p300 plasmid by LipofectamineTM2000 reagent; experimental group 2: incubated with OCT at the final concentration of 10nmol/L for 24 h, 48 h and 72 h, respectively;experimental group 3: transfected with p300 plasmid and incubated with OCT(the final concentration and time were same to those in experimental group 2); control group: no p300 transfection and no OCT incubation.3. Effects of p300 and OCT incubation on cell cycle and proliferation of gastric cancer cells The MTT and flow cytometry were used to observe the effects of p300 and OCT on the proliferation and cell cycle in gastric cancer cells SGC7901 and BGC823.4. Effects of p300 and OCT incubation on the immuno-reactivity of ZAC and ac K14-H3 The fluorescence immunocytochemistry was used to observe the immuno-reactivity(IR) of ZAC and ac K14-H3.5. Statistical analysis All the data were showed as mean ± standard deviation( sx ±) and analyzed with SPSS 17.0 statistical software. P<0.05 was considered as statistically significant.Results1. Identification of eukaryotic expression vector of wild type p300 Agarose gel electrophoresis analysis showed that the length of wild type plasmid p300 DNA fragment was consistent with the expected result. The result of sequencing of wild-type p300 plasmid was consistent with that in Gen Bank.2. Results of MTT In gastric cancer cell SGC7901, the A value of the control group cultured for 24 h, 48 h and 72 h was 0.413 + 0.010, 0.725 + 0.037 and 0.862 +0.012; that of p300 group was 0.271 + 0.010, 0.274 + 0.039 and 0.194 + 0.025; that of OCT group was 0.203 + 0.044, 0.294 + 0.027 and 0.404 + 0.012; that of p300 +OCT was 0.216 + 0.005, 0.229 + 0.017 and 0.153 + 0.011, respectively. In gastric cancer cell BGC823, that of control group was 0.463 + 0.019, 0.782 + 0.032 and0.921 + 0.050; that of p300 group was 0.285 + 0011, 0.230 + 0.028 and 0.165 +0.006; that of OCT group was 0.235 + 0.032, 0.342 + 0.047 and 0.408 + 0.021; that of p300+OCT group was 0.195 + 0.036, 0.184 + 0.023 and 0.116 + 0.018,respectively. Compared with the control group, the cell proliferating ability of the experimental group was significantly decreased(P<0.01), but there was no significant difference between the p300 + OCT group and the OCT group(P > 0.05).The results indicated that both of p300 and OCT could inhibit the proliferation of gastric cancer cells, but there was no synergistic effect between p300 and OCT.3. Results of flow cytometry The results of flow cytometry showed that the number of cells in G0/G1 phase of experimental group was significantly higher than that of the control group(P<0.01). At the same time, that in S phase of experimental group was significantly lower than that of the control group(P< 0.01). However,there was no significant difference between p300 + OCT group and OCT group(P >0.05). The results indicaed that both of p300 and OCT could induce cell cycle arrest in gastric cancer cells, but there was no synergistic effect between p300 and OCT.4. Results of fluorescence immunocytochemistry In gastric cancer cell SGC7901, the average level of ZAC IR in the control group cultured for 24 h, 48 h and 72 h was 0.455 + 0.038, 0.492 + 0.021 and 0.495 + 0.019; that in p300 group was0.622 + 0.026, 0.725 + 0.040 and 0.735 + 0.030; that in OCT group was 0.534 +0.037, 0.629 + 0.039 and 0.754 + 0.058; that in p300 + OCT group was 0.661 +0.021, 0.732 + 0.033 and 0.827 + 0.025, respectively. In gastric cancer cell BGC823,that in the control group was 0.379 + 0.015, 0.346 + 0.013 and 0.356 + 0.018; that in p300 group was 0.576 + 0.021, 0.640 + 0.001 and 0.740 + 0.014; that in OCT group was 0.573 + 0.025, 0.671 + 0.020 and 0.711 + 0.015; that in p300 + OCT was 0.582+ 0.021, 0.610 + 0.048 and 0.829 + 0.010, respectively.The average levels of ZAC IR in the experimental group were significantly higher than those in the control group(P<0.01), but there was no significant difference between the p300 + OCT group and the OCT group(P>0.05). The results indicated that both of p300 and OCT could upregulate the expression of ZAC gene in gastric cancer cells, but there was no synergistic effect between p300 and OCT.In gastric cancer cell SGC7901, the average level of acK14-H3 IR in the control group cultured for 24 h, 48 h and 72 h was 0.479 + 0.015, 0.439 + 0.014 and 0.354 +0.019; that in p300 group was 0.790 + 0.016, 0.831 + 0.020 and 0.489 + 0.031; that in OCT group was 0.562 + 0.037, 0.671 + 0.020 and 0.807 + 0.019; that in p300+OCT group was 0.661 + 0.029, 0.685 + 0.015 and 0.619 + 0.023, respectively.In gastric cancer cell BGC823, that in contral group was 0.416 + 0.025, 0.392 +0.021 and 0.395 + 0.019; that in p300 group was 0.831 + 0.020, 0.752 + 0.032 and0.555 + 0.039; that in OCT group was 0.514 + 0.029, 0.649 + 0.033 and 0.804 +0.059; that in p300 + OCT group was 0.531 0.038, 0.611 + 0.025 and 0.734 + 0.036,respectively. The results indicated that both of p300 and OCT could upregulate the acetylation of histone H3 in gastric cancer cells, but there was no synergistic effect between p300 and OCT.Conclusions1. Both of p300 and OCT can inhibit the proliferation of gastric cancer cells SGC7901 and BGC823, and induce cell cycle arrest, but no synergistic effect between them.2. Both of p300 and OCT can upregulate the expression of ZAC gene in gastric cancer cells BGC823 and SGC7901, but there was no synergistic effect between them.3. Both of p300 and OCT can up regulate the acetylation of histone H3 in gastric cancer cell SGC7901 and BGC823, there was no synergistic effect between them. |
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